A small library of pyrazine amides (including the m-Cl benzylamide, MMV668958) showed promising potency. However, other amides (derived from aliphatic or anilines) were either showed lower potency or were inactive. The RLM of MMV668958 was poor - perhaps due to benzylic oxidation. Alpha-substitution at the benzylic position or constraining the benzylamine into an aminoindane did not improve potency.

A small library of pyrazine amides (including the m-Cl benzylamide, MMV668958) showed promising potency. However, other amides (derived from aliphatic or anilines) were either showed lower potency or were inactive. The RLM of MMV668958 was poor - perhaps due to benzylic oxidation. Alpha-substitution at the benzylic position or constraining the benzylamine into an aminoindane did not improve potency.

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The p-Cl benzanilide MMV670246, although not active against PfNF54, showed good RLM stability (the m-Cl had poor RLM stability) perhaps due to lack of benzylic metabolism. However, its rat PK showed high clearance.

The p-Cl benzanilide MMV670246, although not active against PfNF54, showed good RLM stability (the m-Cl had poor RLM stability) perhaps due to lack of benzylic metabolism. However, its rat PK showed high clearance.

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[[Image:MMV670246.png|thumb|center|400px|MMV670246]]

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[[Image:MMV670246.png|thumb|center|200px|MMV670246]]

Several attempts to make aniline-amides with improved potency against Pfal failed:

Several attempts to make aniline-amides with improved potency against Pfal failed:

Open Source Malaria Series 4: The Triazolopyrazine (TP) Series

Introduction

Preamble

The Triazolopyrazine Series is the newest of the OSM series. It was announced on September 10th 2013 and is sometimes referred to as the TP Series, or OSM Series 4.

Representative Series 4 Compound

The series arises from industrial work that cannot be fully disclosed which was followed by some hit-to-lead work funded directly by MMV and performed by a CRO which can.

A great deal of exploration of the series has been done, with significant diversity in the core and pendant groups. The series includes many potent compounds.

There is evidence from parasite ion regulation assays (below) that these compounds may be PfATP4 inhibitors. Such evidence distinguishes Series 4 from Series 1-3 where there was no experimental evidence for a mechanism of action.

As with everything involved in OSM, suggestions can be given in multiple ways.

The aim of the campaign at the outset was to improve the metabolic stability and the pharmacokinetic properties of this series in rat so as to meet the once-dosing criteria (TCP1) set by MMV. New chemistry directed towards blocking the putative metabolic sites was a major part of the research prior to the data being contributed to OSM.

Notable Points about the TP Series

Seems to have good in vitro HLM and hHEP stability Clint < 8.1 is compatible with 10 nM potency.

RLM remains stubbornly high, particularly for the more potent analogues translating to short half-lives in rat PK.

Series appears to have little polypharmacology or cytotoxicity.

Not so far challenged the hypothesis that rat metabolism may not be a great model for human metabolism for this series.

The series shows activity in Kieran Kirk’s PfATP4 assay which goes away for Pfal inactives in the series.

Concerns about the TP Series

Although dofetilide binding looks weak or nil, the series has shown activity in a patch clamp assay at Essen (1-10 uM) which is quite potent though with a window of >100 fold over Pfal potency.

In Kip Guy’s resistant mutants the picture is mixed, but there is still support for the idea that some members of the series are weaker in the resistant strains. The series has no or weak >>1uM activity against gametocytes, no activity against Winzeler’s Pb liver stage and may have weak activity against ookinetes but the dose-response data has not been completed.

Project Strands of Current Interest

The biggest issue is metabolic stability, as measured in rat in particular. There are few toxicity concerns. Thus possible future directions:

Small scale changes around the side chains, particularly phenethyl to attempt to balance potency and metabolism. Other possibilities: a) N is tolerated in the ring, hasn’t been explored much recently. b) Is 3,4-diF the best substitution pattern? c) Some evidence (eg MMV669848) that the phenethyl side chain can be rigidified, perhaps the iso-indoline of that compound could be improved on with other ring systems and by more optimal substitution of the aromatic benzene ring of the isoindoline. d) The amide MMV670944 is interesting and shows good RLM stability, but many other amides failed to match its potency.

Incorporation of a basic centre to increase volume as a potential fix for half-life. However, this might come at the expense of plasma concentration so would require high potency. Of the 29 compounds with a basic centre only one (MMV670437, below) has a measured potency < 100 nM (actually 44 nM).

MMV670437

More significant structural changes. Of the changes made to the basic skeleton, the most successful might be the recent evaluation of the substitution position changes (e.g., MMV670945), possibly in combination with modifying the disposition of the N atoms in the core. Related compounds have been made by others and it would be wise to incorporate the learnings from these series into any plans to explore this substitution pattern further. The first few compounds look similar in terms of metabolic stability.

SAR

Cycloaliphatic Triazole Substituent

Attempts at lowering the lipophilicity of the TP compounds by replacing the triazole aryl substituent with a cyclo(hetero)aliphatic group, linked either by the heteroatom or otherwise (e.g. piperidine, tetrahydropyran, indoline or isoindoline) lowered the potency against PfNF54, as did an aniline substituent.

Changes to the Triazole Substitution

Core Modification of Pyrazine Ring

Based on an assumption that the pyrazine moiety of the TP could undergo AO metabolism at positions alpha- to the nitrogen a few compounds were made with different R groups (Cl, Me, NH2, NEt2). However, all these compounds lost potency against PfNF54.

Changes to the Pyrazine Substitution

Core Modification of Triazole Ring

Two compounds based on imadazopyrazines were made (MMV669846 and MMV670250, below). Both showed reduced potency against PfNF54 vs. the corresponding TP compound. The RLM stability of one was found to be poor. Approx 20 structures were made with variations to the 6,5 core system. MMV669846 was the most potent. As most of the analogues were >1 μM potency, fewer were tested in RLM (quite a few in HLM). Of the 4 tested in RLM, the greatest stability had a Clint of 109, (HLM 9.5), several had HLM Clint 8 or less, particularly after moving or removing the N from the pyrazine ring.

Core Modification of Triazine Ring

(Note - original briefing document contained two entries for MMV669846 at this point with different potencies - need check of original spreadsheet, to verify the above is correct)

Transposing the Pyrazine side-chain

The side-chain on the pyrazine ring was shifted to the adjacent carbon. The chain length was varied (n= 0,1,2) and linked through either O or N.

A small library of pyrazine amides (including the m-Cl benzylamide, MMV668958) showed promising potency. However, other amides (derived from aliphatic or anilines) were either showed lower potency or were inactive. The RLM of MMV668958 was poor - perhaps due to benzylic oxidation. Alpha-substitution at the benzylic position or constraining the benzylamine into an aminoindane did not improve potency.

MMV668958

The p-Cl benzanilide MMV670246, although not active against PfNF54, showed good RLM stability (the m-Cl had poor RLM stability) perhaps due to lack of benzylic metabolism. However, its rat PK showed high clearance.

MMV670246

Several attempts to make aniline-amides with improved potency against Pfal failed:

Loading the aniline ring with lipophilic substituents marginally improved potency but led to poor RLM stability.

Considering that the benzylic position in the phenethyl side-chain is prone to metabolic oxidation, several compounds having mono- and di-substitution in the benzylic position were made:

The phenyethyl chain was replaced by an aromatic group in an attempt to mitigate the potential metabolism of the ethyl chain. Several compounds with a phenol substituent on the pyrazine ring were made. Some substituted phenolates were metabolically more stable in-vitro as well as in-vivo in rat, although with reduced potency against Pfal.

IMAGE

A 2-naphthol substituent on the pyrazine ring showed reasonable potency against Pfal (IC50 114 nM) but suffered from poor RLM stability. Several hetero-analogs of 2-naphthol e.g. indole, indazole, quinoline, chroman, benzisoxazole, quinazoline, etc were made but all lost potency against the parasite.

Pharmacokinetics

Only one compound in this series first made at the CRO was measured in rat PK and that was the relatively weak amide MMV670246. The curve is shown for oral & IV legs & parameters are below.

IMAGE + chart

One of the compounds with better in vitro balance is MMV670652 with potency at 17nM, HLM Clint < 8 ul/min/mg and RLM Clint at 30 ul/min/mg. This compound has not been in rat PK. Additionally it may be possible to improve potency by synthesis of the more potent enantiomer.

IMAGE

Other Observations

As would be expected HLM vs RLM shows a general correlation with approx 4-fold shift on average. However, for most of the more potent analogues, this increases to over 10-fold. The figure below shows the 4 sub 30nM compounds with HLM & RLM measured.

Potential next steps

The series has good potency and in vivo efficacy with few toxicity concerns. The biggest issue is metabolic stability, as measured in rat in particular. Some possible future directions include:

Small scale changes around the side chains, particularly phenethyl to attempt to balance potency and metabolism

N is tolerated in the ring, hasn’t been explored much recently

Is 3,4-diF the best substitution pattern ?

Some evidence (eg. MMV669848) that the phenethyl side chain can be rigidified, perhaps the iso-indoline of that compound could be improved on with other ring systems and by more optimal substitution of the aromatic benzene ring of the isoindoline.

The amide MMV670944 is interesting and shows good RLM stability, but many other amides failed to match its potency

Incorporation of a basic centre to increase volume as a potential fix for half-life. However, this might come at the expense of plasma concentration so would require high potency. In addition of the 29 compounds with a basic centre only one has a measured potency < 100nM.

IMAGE

More significant structural changes. Of the changes made to the basic skeleton, the most successful might be the recent evaluation of the substitution position changes (eg MMV670945), possibly in combination with modifying the disposition of the N atoms in the core. Related compounds have been made by others and it would be wise to incorporate the learnings from these series into any plans to explore this substitution pattern further. The first few compounds look similar in terms of metabolic stability.

What Can the Community Do Now?

Those wishing to contribute to OSM Series 4 should in the first instance read the briefing document. The first question is: Which 10 compounds ought to be made in Sept/Oct 2013 for evaluation?

Resources Needed: Chemists to either make new molecules, or help obtain existing compounds that might be relevant to this series.

Current Aims

Current Questions for the Community

Starting-point questions are listed at the end of the briefing document. These translate into the initial question of Which 10 compounds ought to be made first? (also asked here and Does anyone possess compounds that could be relevant to this series already? (asked here and there is a separate section for results below).

Prior Knowledge of the Series

Potency of Hits and Analogs

(Detail will be placed here, but for the moment please browse the briefing document).

Possible PfATP4 Activity Deduced from Parasite Ion Regulation Assays

The following five compounds were evaluated in parasite ion regulation assays in the Kirk Laboratory; the hypothesis is that PfATP4 is a Na+ ATPase that exports Na+ and imports H+ (or equivalent) and that the effects of the compounds on Na+ concentration and pH are attributable to inhibition of this activity. Structures, potency, metabolism/solubility and raw PfATP4 assay data are here.